The disclosed invention is in the general field of nucleic acid amplification and detection, and specifically in the field of amplification through continuous flow mechanisms.
Continuous-flow polymerase chain reaction (CF-PCR) is an amplification technique in which a single fluidic channel is heated with spatial temperature variations such that a flowing sample experiences the thermal cycling required to induce amplification. This heating method reduces the thermal load to only that of the sample being amplified. By excluding the substrate from the thermal cycling, lower energy consumption and faster cycling can be achieved. This has been demonstrated with a variety of thermocycling techniques, including infrared (IR) heated PCR systems (Roper, 2007), shuttle PCR devices (Chiou, 2001), and CF-PCR instrumentation. CF-PCR was first demonstrated in a microfluidic device Kopp and coworkers (Kopp, 1998). This foundational design consisted of a microfluidic serpentine channel embedded within a glass substrate. Three heaters were fixed to the chip to produce distinct thermal zones through which the fluid would pass. Other researchers have continued to improve the operation of this original 20-cycle device. Li and coworkers (Li, 2006) built a device whose 20-cycle serpentine microchannel was narrower in the regions between the three temperature zones, thus reducing the inter-temperature transition time. Schneegass and coworkers (Schneegass, 2001) built a 25-cycle device from silicon and glass. The device included integrated heaters and temperature sensors which were fabricated on-chip using IC manufacturing technology. Fukuba and coworkers (Fukuba, 2004) were able to automate the operation of a 30-cycle device using miniature pumps and valves. Sun and coworkers (Sun, 2002) have developed a 30-cycle CF-PCR device with integrated ITO heaters (indium tin oxide), thus making the device optically transparent. Obeid and coworkers (Obeid, 2003b) presented a device capable of the reverse transcription of RNA prior to its amplification in a 40-cycle serpentine channel (RT-PCR). The device was fabricated with outlets at cycle numbers 20, 25, 30, 35, and the full 40. In addition, the researchers were able demonstrate amplification with plug flow, thus reducing the amplification volume to only 2 μl per amplified sample. While these previous projects do represent significant improvements for CF-PCR, they all implement the original heating scheme: multiple zones of distinct temperatures, placed in parallel, through which a serpentine channel repeatedly passes. An alternative layout was presented by Hashimoto and coworkers (Hashimoto, 2004), who developed a device in which the isothermal zones were separated into the four quadrants of a rectangular substrate. By fabricating a 20-loop spiral microchannel which passes repeatedly through each zone, the flowing fluid was able to experience the required thermocycling.
Integration of these continuous-flow amplification systems is currently being accomplished by several groups. Obeid and coworkers (Obeid, 2003a) have combined a continuous-flow RT-PCR with an laser-induced fluorescence (LIF) detection system. Nakayama and coworkers (Nakayama, 2006) have demonstrated real-time amplification detection using TaqMan technology. Wang and coworkers (Wang, 2006) have used a quadrant heating/spiral channel CF-PCR device as an amplification module within a Sanger sequencing system. In addition, other technologies are being developed that could potentially be included to form a complete “Lab-on-a-chip”, such as continuous-flow DNA extraction (Cao, 2006) and sample mixing (Garstecki, 2006).
The further miniaturization and simplification of the CF-PCR device is critical for this technology to compete against other micro-PCR methods. Researchers have shown that by including insulating features in the fabricated devices, better thermal separation between the several temperature zones is possible (Hashimoto, 2004; Schneegass, 2001; Yang, 2005). While this allows for a reduction in the spacing between the heaters, thermal “cross-talk” ultimately limits the proximity of the isothermal regions (Li, 2006). Thus, the need for multiple isolated temperature zones greatly complicates further reduction in the CF-PCR footprint.